Tri-Functional CRISPR Screen Reveals Overexpression of QDR2 and QDR3 Transporters Increase Fumaric Acid Production in Kluyveromyces marxianus

Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7 , encoding a subunit of the mitochondrial F ₁ F ₀ ATP synthase, and overexpression of QDR2 and QDR3 , two previously uncharacterized members of the 12-spanner H⁺ antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a Δ FUM1 background strain improved titers of fumaric acid production from 0.26 g L ⁻¹ to 2.16 g L ⁻¹ . Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus .